In Vivo Identification of an HLA-G Complex as Ubiquitinated Protein Circulating in Exosomes

Eur J Immunol. 2013 Jul;43(7):1933-9. doi: 10.1002/eji.201343318. Epub 2013 May 17.

Abstract

The nonclassical human leukocyte antigen-G (HLA-G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA-G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA-G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA-G by ELISA. Complexed HLA-G was detected in exosomes, which indicates an intracellular origin of these forms. 2D-PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA-G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA-G complexes. Immunoblot analysis of exudates and exosomes with an anti-ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA-G. HLA-G ubiquitination could be reproduced in vitro in HLA-G1-transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA-G forms in vivo that open a new perspective in the study of HLA-G function and analysis.

Keywords: Exosome; Exudates; HLA-G; Molecular immunology; Ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ascites / immunology
  • Blotting, Western
  • Electrophoresis, Gel, Two-Dimensional
  • Enzyme-Linked Immunosorbent Assay
  • Exosomes / chemistry
  • Exosomes / immunology
  • Exosomes / metabolism*
  • HLA-G Antigens / immunology
  • HLA-G Antigens / isolation & purification
  • HLA-G Antigens / metabolism*
  • Humans
  • Immunoprecipitation
  • Pleural Effusion / immunology
  • Ubiquitinated Proteins / immunology
  • Ubiquitinated Proteins / isolation & purification
  • Ubiquitinated Proteins / metabolism
  • Ubiquitination

Substances

  • HLA-G Antigens
  • Ubiquitinated Proteins